The human pathogen Vibrio cholerae uses quorum sensing which involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers, to regulate virulence gene expression in response to changes in cell-population density and species complexity. Two V. cholerae autoinducer-detection systems, one for intra-species and one for inter-species communication, are currently known. The long term goal of this application is to understand the molecular mechanisms underlying intra-species and inter-species quorum-sensing signaling in this bacterium. The unique information encoded in each extracellular autoinducer is translated internally into discrete levels of the phosphorylated response regulator LuxO, which determines the final output of the V. cholerae quorum-sensing system. Due to the instability of aspartyl phosphates, in vivo levels of phosphorylated bacterial response regulators have never been directly determined. To understand the signal-response input-output relationship in the V. cholerae quorum-sensing circuit, my first aim is to develop methodology for the quantitative measurement of phospho-LuxO levels in V. cholerae cells. Integration of the information contained in the two different autoinducers into a single regulatory network could allow the V. cholerae quorum-sensing system to function as a coincidence detector. Alternatively, this circuit may facilitate distinct gene regulation by any or all combinations of the two autoinducers. My second aim is focused on distinguishing between these two possibilities by investigating the effect of each autoinducer on specific target gene expression. Measurement of the temporal expression dynamics of selected target genes will be performed to establish whether the autoinducers function together or if there is an ordered gene expression hierarchy in response to different autoinducer combinations. Results from these studies will fully define the role of each regulatory element in the quorum-sensing signaling circuit. The third aim of this application is to identify novel chemical signal communication pathways that exist between V. cholerae and other bacterial species. Inter-species communication likely enables V. cholerae to adapt, compete, and survive in complex environments such as contaminated water and the human intestine where many other bacterial species are present. The components involved in such inter-species communication pathways will be identified and characterized. Together, these studies will provide insight into the molecular architecture of V. cholerae intra- and inter-species communication networks. Practically, results from this work could lead to the development of anti-quorum-sensing therapies for use as alternatives to traditional antibiotics.